We propose to continue our study of extracellular matrix vesicles which appear to play an initiating role in calcification of cartilage, bone and dentin. These submicroscopic, extracellular membranous vesicles were first identified and described in the matrix of calcifying cartilage. Subsequently, we isolated matrix vesicles from normal and rachitic growth cartilage and demonstrated that they contain much phosphatase, especially alkaline phosphatase, ATPase and inorganic PPiase, and are enriched in lipids, especially acidic phospholipids. All of these observations have been confirmed by other investigators. Two major questions require further study: 1) the mechanism of vesicles calcification in rachitic cartilage and embryonic bone. Procedures have already been worked out for culture and assay of calcium uptake by both of these tissues. Controlling factors in vesicle calcification will be identified by altering culture conditions, and then tested for their ability to stimulate calcium uptake by isolated matrix vesicles. We shall also attempt to develop new and less damaging methods for vesicle isolation. In order to study the biogenesis of matrix vesicles, we propose using autoradiography to observe uptake into precursor cells and release into the matrix of membrane lipid precursor, (such as H3-glycerol.) This is a fundamental study which is expected to provide new insight and control over the mechanism by which cells initiate calcification. New knowledge of this process can apply to a broad range of topics including specific disease states.